The sarcoplasmic reticulum (SR) of skeletal muscle is a heterogeneous membrane system consisting of several protein components embedded in a lipid bilayer. Its primary function is associated with an ATP-dependent active transport of calcium to and from calcium binding sites of the contractile proteins. The molecular mechanism of this calcium transport process remains unclear. There is little information about the side chains of amino acid residues at the active site of the skeletal muscle ATPase protein of SR that may participate directly in the bond forming and breaking steps with substrate ATP, in the binding of this nucleotide or in the binding of phospholipids. Affinity labeling with ATP analogs such as 2(4-azido-2- nitrophenyl)amino)ethyl triphosphate and 5'-para- fluorosulfonylbenzoyladenosine or azido-arylphospholipids will be studied. The ATP analogs contain reactive groups substituted in different regions of the nucleotide and they provide a means for determining the ATP binding site environment. To constrain these affinity labels and localize their specificity experiments will be performed in the presence of bifunctional reagents. Reactive groups of the ATPase protein (thiol or tyrosyl), in close proximity to each other, can be modified by reagents such as para-phenylenedimaleimide or 1,5-difluoro-2, 4-dinitrobenzene in a bifunctional manner. This may lead to the trapping of the ATP analogs within the ATPase protein. Concurrently, the modified ATPase will be assayed for calcium-activated ATPase activity and its ability to support calcium uptake will be determined in reconstitution experiments. Characterization of the active site and phospholipid regions of the ATPase protein will add to our knowledge of the mechanism controlling contractile function in skeletal muscle.